Cytokines comprise a variety of secreted proteins that regulate cell growth and differentiation. These factors dramatically influence immune and inflammatory responses. Furthermore, cytokines are critical for lymphoid development, homeostasis, tolerance, and memory. Thus understanding the molecular basis of this regulation is likely to provide important insights on the pathogenesis of immune-mediated disease as well as offer new therapeutic targets. We cloned a kinase, Jak3 that is responsible for signaling by a class of cytokines that bind the common gamma chain, gc (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21). Mutation of gc or Jak3 results in severe combined immunodeficiency (SCID). A major objective of the laboratory continues to be the understanding the structure and function of Jak3, in vitro and in vivo. One aspect of this project is the identification of new patients with Jak3 mutations. We recently identified one family in which different members exhibited a range of presentations from profound immunodeficiency and severe autoimmunity to being relatively unaffected. We further showed that these Jak3 mutations were associated with oligoclonal expansion of activated T lymphocytes with poor expression of the pro-apoptotic molecule Fas ligand. This suggests that Jak3, likely via IL-2 activation, is essential in humans for lymphoid homeostasis. The data also indicates that the range of clinical presentations associated with Jak3 mutations is much broader than previously appreciated. We also utilized patient mutations to provide new insights into the structure of Jak3. We identified three patient-derived mutations in the amino-terminal FERM (band 4.1, ezrin, radixin and moesin domain). Through the study of these patients, we showed that FERM mutations inhibit Jak/receptor association and inhibit Jak3 catalytic activity. We further showed that the likely explanation for these results was that the FERM and kinase domains associate and that this association positively regulates catalytic activity. Jaks associate with a variety of additional proteins that regulate their activity. We found that the adapter molecule SH2-B beta binds to Jak1, Jak2 and Jak3, but only regulates the catalytic activity of Jak2. In contrast to SH2-B beta, the related adapter APS decreased tyrosyl phosphorylation of GH-stimulated JAK2 as well as Stat5B, a substrate of JAK2. APS also decreased tyrosyl phosphorylation of JAK1, but did not affect the activity or tyrosyl phosphorylation of JAK3, though APS binds all three JAKs. We had previously identified the docking protein, Gab2, as a Jak3 substrate. In extension of these studies, we have now shown that Gab2 is also an important adapter molecule involved in Fc receptor (FcR)-mediated signaling. We showed that is a unique substrate in that the adapter molecule LAT and the kinases, Syk and Lyn are not required for its phosphorylation. The second major area of investigation is the control of helper T (Th) cell differentiation and the regulation of cell-mediated immunity. Several factors regulate this process including the cytokine IL-12, which activates the transcription factor Stat4. We previously demonstrated that IL-12 activates the Janus kinases, Tyk2 and Jak2 and the transcription factor Stat4. The IL-12R comprises two subunits IL-12R beta 1 and IL-12R beta 2 and we previously demonstrated that the latter is essential for Th1 differentiation. In a collaborative study, we identified the phosphorylation sites of IL-12R beta 2 for recruiting Stat4 to the receptor. These sites are essential for IL-12- mediation induction of IFN-gamma and Th1 differentiation. Furthermore, transgenic expression of IL-12R beta 2 was not sufficient to drive Th1 differentiation in the absence of Stat4. We have also investigated other aspects of IL-12 signaling, including the ability of the IL-12R to couple to p38 MAPK activation and have identified Ser721 of Stat4 as a substrate of this kinase. By mutational analysis we demonstrated that this modification is important in control of transcriptional regulation. By reconstitution of Stat4 deficient lymphocytes with a Ser721Ala mutant of Stat4, we found that phosphorylation of this site is essential for normal IFN-gamma production and Th1 differentiation. T-bet is a newly described transcription factor that also promotes Th1 differentiation and IFN-gamma production. We investigated the regulation of T-bet and showed that it is rapidly induced by IFN-gamma itself. That is, naive T cells express little T-bet whereas TCR occupancy with IFN-gamma synergistically upregulate T-bet. IFN-gamma regulation of T-bet is dependent upon Stat1 but is independent of Stat4. Thus, we propose a new model of Th1-differentiation in which IFN-gamma regulates its own production by induction of T-bet; T-bet/IFN-gamma cooperate in a feed-forward mechanism of Th1 regulation. It is now appreciated that dendritic cells (DC) and other antigen presenting cells (APCs) regulate Th differentiation and we demonstrated that the expression of Stat4 in dendritic cells is required for IFN-gamma production and is involved in autocrine IL-12 responsiveness. Based on these findings, we next investigated the expression of T-bet in DC and macrophages. We demonstrated that IFN-gamma induces expression of T-bet in these APCs. We propose that the regulation of expression of "Th1"-expressed transcription factors, like T-bet and Stat4, control IFN-gamma production in APCs and thus serve to regulate Th1 differentiation. We have termed this the "jump start" model of cell-mediated immunity to denote the ability of DC to initiate Th1 differentiation by providing IFN-gamma. Type I interferons, IFN-alpha/beta, are critical in host defense against pathogens due to their potent antiviral and immunoregulatory functions. Some of these effects are paradoxical and depending on the conditions, they can either enhance or inhibit IFN-gamma production. We showed that type I IFNs activate Stat4, and that Stat4 is necessary for endogenous IFN-gamma production dependent on CD8 T cells during viral infections. IFN-alpha/beta also synergize with T cell receptor signals for the production of IFN-gamma. In contrast, Stat1 negatively regulates IFN-alpha/beta induction of IFN-gamma. These results establish a mechanism by which the type I IFN effects for shaping innate to adaptive immunity are regulated by accessibility to intracellular signaling molecules.In effort to better understand target genes activated by cytokines, we have utilized microarray technology. We identified a gene we term Cybr, cytohesin binder and regulator that is induced by IL-12 and preferential expressed in Th1 cells. It associates with guanine nucleotide exchange protein, cytohesin and promotes the GTPase activity of ARFs. Given the therapeutic utility of glucocorticoids, we also employed microarray technology to investigate the effects of glucocorticoids on activated T-cells. A number of important genes were identified, but we were particularly intrigued, that one of the most highly glucocorticoid-inducible genes is the interleukin-7 receptor (IL-7R). We demonstrated that glucocorticoids can have the unexpected function of enhancing lymphoid survival by upregulating IL-7R. Finally, in collaboration with the Genetics Section of ARB, we showed that the disorder, NOMID (neonatal onset multisystem inflammatory disease) is due to mutations of CIAS1 and is associated with marked increases in cytokine production.